Application of compound in preparation of medicine for treating psoriasis

Abstract

The invention discloses an application of a compound in preparation of a medicine for treating psoriasis, and particularly discloses a new application of lefamulin or retapamulin in preparation of a medicine for treating psoriasis, experiments prove that lefamulin or retapamulin has a remarkable curative effect on treating psoriasis, takes effect quickly and has small toxic and side effects, and the lefamulin or retapamulin has no side effects on psoriasis. The medicine for treating psoriasis is safe, efficient, stable, simple in preparation process, suitable for industrial production and easy to popularize. The invention provides a new medicine source for treating psoriasis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to the application of a compound in the preparation of a drug for treating chronic inflammatory skin diseases, and in particular to the application of a compound in the preparation of a drug for treating psoriasis. Background Art

[0002] Psoriasis is a common clinical chronic inflammatory and proliferative skin disease with erythematous scaling and epidermal thickening as the main clinical manifestations. It can affect multiple tissues such as the patient's skin, mucous membranes and joints. Psoriasis has a long course, is difficult to cure, and is prone to recurrence. Most people suffer from it for life. The itching and pain it causes have a serious impact on the patient's physical and mental health, and also bring a huge economic burden to the patient's family and the entire society. The cause and pathogenesis of psoriasis are complex. Genetics, infection, immunity, metabolic disorders, mental, environmental and other factors are closely related to the disease. Various factors interact with each other to form a network, which also leads to the multi-center onset of psoriasis and the complexity of psoriasis prevention and treatment.

[0003] There is currently no effective cure for psoriasis. Commonly used clinical treatment measures can be divided into physical therapy, local drug therapy and systemic drug therapy. Physical therapy and local drug therapy are suitable for patients with milder symptoms, while moderate and severe patients require systemic drug therapy. Traditional local treatment for psoriasis mainly uses topical corticosteroids, but long-term use of topical corticosteroids may cause a series of adverse reactions such as skin atrophy and hirsutism.

[0004] In recent years, progress has been made in the development of monoclonal antibody drugs targeting key inflammatory factors in the pathogenesis of psoriasis, such as tumor necrosis factor alpha (TNF-α) and interleukin 17 (IL-17). However, drugs such as adalimumab (TNF-α monoclonal antibody) and secukinumab (IL-17 monoclonal antibody) have many inherent defects, such as high price, inability to be taken orally or topically, and serious side effects caused by long-term use.

[0005] Therefore, the development of drugs for psoriasis is one of the major issues that need to be solved urgently in current experimental research and clinical practice. Studies in recent years have shown that small molecule drugs have many advantages such as low cost and flexible administration methods. Therefore, finding and developing small molecule drugs for psoriasis is a hot topic in current research.

[0006] Retapamulin, molecular formula C 30 H 46 NO4S, the structural formula is shown in formula (I),

[0007]

[0008] It is a local antibiotic, often used as an external antibacterial drug. Studies on this compound have found that it has strong antibacterial activity against Staphylococcus aureus and Streptococcus pyogenes. There have been no reports on its use in the treatment of psoriasis so far.

[0009] Lefamulin, molecular formula C 28 H 45 NO5S, the structural formula is shown in formula (II),

[0010]

[0011] It is a pleuromutilin antibiotic. Studies on this compound have found that it is mainly used to treat community-acquired bacterial pneumonia (CABP) caused by sensitive microorganisms. There have been no reports on its use in the treatment of psoriasis. Summary of the invention

[0012] In view of the above problems, one of the objects of the present invention is to provide a class of compounds for use in the preparation of drugs for treating psoriasis. Such compounds are lefamulin or retapamulin or their derivatives. Such compounds are non-hormonal compounds and can be administered externally, which significantly reduces side effects and has less impact on the whole body, and can be better used in drugs for the treatment of psoriasis.

[0013] The present invention mainly finds that lefamulin and retapamulin have a significant improvement effect on the imiquimod-induced mouse psoriasis-like model, and the results of immunoblotting experiments indicate that lefamulin and retapamulin have antagonistic effects on TNF-α and its pathway, so they can be used in the treatment of psoriasis.

[0014] The present invention uses imiquimod (IMQ) to model psoriasis in mice, psoriasis-like dermatitis appears on the back skin of mice, and retapamulin and lefamulin topical medicines are prepared, and lefamulin and retapamulin topical medicines are used to treat psoriasis-like dermatitis, and it is found that both drugs can treat imiquimod-induced psoriasis-like dermatitis. In addition, the present invention uses TNF-α to model cell death in L929 cell line, and then conducts experiments. The experimental results show that the administration of retapamulin and lefamulin by the present invention can significantly inhibit TNF-α-induced L929 cell death and alleviate IMQ-induced skin erythema and scaling. Chemical proteomics is an important tool for discovering the action targets of small molecule drugs. Experiments have found that lefamulin interacts with heterogeneous nuclear ribonucleoprotein U (hnRNP-U). Using siRNA to knock down hnRNP-U can inhibit TNF-α-induced cell death. The results of immunoblotting suggested that lefamulin could inhibit the expression of hnRNP-U in HaCaT cells. In immunohistochemistry, hnRNP-U was found to be highly expressed in psoriatic-like lesions and psoriasis lesions in mice, indicating that hnRNP-U was associated with the activity of psoriasis. The above results showed that retapamulin and lefamulin could inhibit the TNF-α pathway through hnRNP-U and thus improve the symptoms of IMQ-induced psoriasis. Moreover, the small molecule can be administered externally, so the side effects are relatively small and the impact on the whole body is also small. Therefore, it can be used in the treatment of psoriasis.

[0015] To achieve the above objectives, the present invention provides the following technical solutions: On the one hand, the present invention provides an application of a compound in the preparation of a drug for treating psoriasis, wherein the compound is Retapamulin or Lefamulin or a derivative thereof.

[0016] In some embodiments, the derivative is in the form of a salt and / or a solvate.

[0017] In some embodiments, the derivative of lefamulin is in the form of lefamulin acetate or lefamulin hydrochloride.

[0018] In some embodiments, the psoriasis is psoriasis vulgaris.

[0019] In some embodiments, the medicament contains a therapeutically effective amount of lefamulin or retapamulin and a pharmaceutically acceptable carrier.

[0020] In some embodiments, the therapeutically effective amount is 1% to 10%, specifically, the therapeutically effective amount is 1% to 9%, 2% to 9%, 3% to 9%, 4% to 9%, 5% to 9%, 6% to 9%, 7% to 9%, 8% to 9%, 1% to 8%, 2% to 8%, 3% to 8%, 4% to 8%, 5% to 8%, 6% to 8%, 7% to 8%, 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, 6% to 7%, 1% to 6%, 2% to 6%, 3% to 6%, 4% to 6%, 5% to 6%, 1% to 5%, 2% to 5%, 3% to 5%, 4% to 5%, 1% to 4%, 2% to 4%, 3% to 4%, 1% to 3%, 2% to 3%.

[0021] In some embodiments, the pharmaceutically acceptable carrier includes one or more of a diluent, a solubilizer, a co-solvent, a disintegrant, a dispersant, a lubricant, a flavoring agent, an antioxidant, a binder, an absorbent, a wetting agent, a buffer, and a cross-linking agent.

[0022] In some embodiments, the drug is formulated into a pharmaceutically acceptable dosage form.

[0023] In some embodiments, the dosage form includes pills, tablets, powders, capsules, granules, powders, pellets, drops, patches, tinctures, pastes, lotions, sprays, injections, suspensions, creams, ointments, gels, and suppositories.

[0024] In some embodiments, the drug is a topical drug.

[0025] In some embodiments, the therapeutically effective amount of lefamulin or retapamulin is 1% to 10%, specifically, the therapeutically effective amount is 1% to 9%, 2% to 9%, 3% to 9%, 4% to 9%, 5% to 9%, 6% to 9%, 7% to 9%, 8% to 9%, 1% to 8%, 2% to 8%, 3% to 8%, 4% to 8%, 5% to 8%, 6% to 8%, 7% to 8%, 1% to 7%, 2% to 7%, 3% to 7%, 4% to 7%, 5% to 7%, 6% to 7%, 1% to 6%, 2% to 6%, 3% to 6%, 4% to 6%, 5% to 6%, 1% to 5%, 2% to 5%, 3% to 5%, 4% to 5%, 1% to 4%, 2% to 4%, 3% to 4%, 1% to 3%, 2% to 3%, specifically, the therapeutically effective amount is 5%.

[0026] In some embodiments, the topical medication is in the form of a spray, an aerosol, a patch, a tincture, a paste, a lotion, a cream, a cream, or an ointment.

[0027] Another aspect of the present invention provides an ointment for treating psoriasis, wherein the ointment comprises lefamulin or retapamulin,

[0028] Wherein, the structural formula of lefamulin is as follows:

[0029]

[0030] The structural formula of retapamulin is shown below:

[0031]

[0032] In some embodiments, the ointment is composed of:

[0033]

[0034] In some embodiments, in the ointment of the present invention, the amount of lefamulin or retapamulin is 0.25 g to 0.75 g, specifically, the amount of lefamulin or retapamulin is 0.25 g to 0.75 g, 0.25 g to 0.70 g, 0.25 g to 0.65 g, 0.25 g to 0.60 g, 0.25 g to 0.55 g, 0.25 g to 0.50 g, 0.25 g to 0.45 g , 0.25g to 0.40g, 0.25g to 0.35g, 0.30g to 0.75g, 0.30g to 0.70g, 0.30g to 0.65g, 0.30g to 0.60g, 0.30g to 0.55g, 0.30g to 0.50g, 0.30g to 0.45g, 0.30g to 0.40g, 0.35g to 0.75g, 0.35g to 0.70g, 0.3 5g to 0.65g, 0.35g to 0.60g, 0.35g to 0.55g, 0.35g to 0.50g, 0.35g to 0.45g, 0.40g to 0.75g, 0.40g to 0.70g, 0.40g to 0.65g, 0.40g to 0.60g, 0.40g to 0.55g, 0.40g to 0.50g, 0.45g to 0.75g, 0.45g to 0 .70g, 0.45g to 0.65g, 0.45g to 0.60g, 0.45g to 0.55g, 0.50g to 0.75g, 0.50g to 0.70g, 0.50g to 0.65g, 0.50g to 0.60g, 0.50g to 0.75g, 0.55g to 0.70g, 0.55g to 0.65g, 0.60g to 0.75g, 0.60g to 0.70g.

[0035] In some embodiments, in the ointment of the present invention, the amount of the polyoxyethylene stearate is 2.0 to 3.0 g, specifically, the amount of the polyoxyethylene stearate is 2.0 to 3.0 g, 2.0 to 2.5 g, or 2.5 to 3.0 g.

[0036] In some embodiments, in the ointment of the present invention, the amount of propylene glycol is 2.0 to 3.0 g, specifically, the amount of propylene glycol is 2.0 to 3.0 g, 2.0 to 2.5 g, or 2.5 to 3.0 g.

[0037] In some embodiments, in the ointment of the present invention, the amount of the white vaseline is 4.0 to 5.0 g, specifically, the amount of the white vaseline is 4.0 to 5.0 g, 4.0 to 4.5 g, or 4.5 to 5.0 g.

[0038] The present invention also provides a use of a compound in the preparation of a drug for inhibiting the TNF-α pathway by interacting with heterogeneous nuclear ribonucleoprotein U (hnRNP-U), wherein the compound is lefamulin, retapamulin or a derivative thereof,

[0039] Wherein, the structural formula of lefamulin is as follows:

[0040]

[0041] The structural formula of retapamulin is shown below:

[0042]

[0043] Unless otherwise indicated, the term "therapeutically effective amount" as used herein is the amount of a drug required to produce an effective effect; "therapeutically effective amount" is adjustable and variable, and can be determined by the attending physician, taking into account factors such as the specific circumstances of the subject being treated, the duration of treatment, the severity of the disease, and reasonable medical judgment, and can be determined mathematically based on the results of animal experiments to determine the therapeutically effective amount of the drug to be administered to humans. The therapeutically effective amount of the drug to be administered for treatment can be easily determined by those skilled in the art, and the optimal dose will vary with the specific compound used, the mode of administration, the formulation specifications, and the progression of disease symptoms. In addition, due to factors related to the specific subject to be treated, including the subject's age, weight, diet, and administration time, the dose needs to be adjusted to an appropriate treatment level.

[0044] The present invention has at least the following beneficial effects: (1) Retapamulin and Lefamulin can improve the symptoms of psoriasis vulgaris. They are non-hormonal compounds and can be administered externally. External administration can significantly reduce side effects and have less impact on the whole body, so they can be better used in the treatment of psoriasis; (2) The present invention has discovered new medicinal values ​​for the known small molecule compounds Retapamulin and Lefamulin, and used them to treat psoriasis, thereby opening up a new application field for the application of Retapamulin and Lefamulin; (3) The pharmacological effects of Retapamulin and Lefamulin of the present invention are strong. Cell death modeling is carried out in the L929 cell line using TNF-α. TNF-α The induced L929 cell death effect was inhibited by lefamulin and retapamulin, and the inhibition efficiency was dose-dependent; (4) After adding photocrosslinking groups to lefamulin, it was found through chemical proteomics screening that hnRNP-U can interact with lefamulin. In HaCaT cells, co-incubation with lefamulin found that the expression of hnRNP-U in HaCaT cells decreased, that is, lefamulin can inhibit the TNF-α pathway through hnRNP-U; (5) In psoriasis-like lesions of mice and lesions of psoriasis patients, it was found that the expression level of hnRNP-U was correlated with the severity of the disease, indicating that lefamulin can achieve the purpose of treating psoriasis by inhibiting hnRNP-U. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Figures 2A and 2B are HE staining images of the changes in the back skin and skin lesions of mice in Example 2A and Example 2B, wherein A is the HE staining image of the changes in the back skin and skin lesions of mice in Example 2A, wherein the left side is the back skin changes image, and the right side is the skin lesion HE staining image; B is the HE staining image of the changes in the back skin and skin lesions of mice in Example 2B, wherein the left side is the back skin changes image, and the right side is the skin lesion HE staining image;

[0046] Figure 2A This is a statistical diagram of the inhibitory effect of lefamulin on TNF-α-induced L929 cell death in Example 3A;

[0047] Figure 2B This is a statistical diagram of the inhibitory effect of retapamulin on TNF-α-induced L929 cell death in Example 3B;

[0048] Figure 3A The molecular structure formula of lefamulin with added photo-crosslinking group (lef-probe) in Example 4;

[0049] Figure 3B This is a statistical diagram of the inhibitory effect of Lef-probe on TNF-α-induced L929 cell death in Example 4;

[0050] Figure 3CThis is a schematic diagram of the grouping state of the proteomic screening of lefamulin protein substrates in Example 4;

[0051] Figure 3D This is a schematic diagram of three repeated experiments in Example 4 to screen potential protein substrates from L929 and HaCaT cells;

[0052] Figure 3E This is a graph showing the ratio of proteins interacting with lefamulin identified in L929 and HaCaT cells in Example 4;

[0053] Figure 4A This is an immunoblot image of the use of Lefamulin to inhibit the expression of hnRNP-U in HaCaT cells in Example 5;

[0054] Figure 4B This is a statistical diagram showing the reduction in the function of lefamulin in inhibiting TNF-α-induced L929 cell death in Example 5;

[0055] Figure 5A This is the observation picture of hnRNP-U immunohistochemical staining of mouse skin in Example 6;

[0056] Figure 5B Statistical graph of hnRNP-U immunohistochemical staining of mouse skin in different treatment groups;

[0057] Figure 5C This is the observation picture of hnRNP-U immunohistochemical staining of skin lesions in patients with psoriasis. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] The beneficial effects of the drug of the present invention are further described below through test examples, which include the efficacy test of the present invention.

[0060] Example 1 Preparation of Lefamulin Ointment and Retapamulin Ointment

[0061] Example 1A Preparation of Lefamulin Ointment

[0062] 1A) Prepare the ingredients according to the following formula:

[0063]

[0064] 2A) Preparation of Lefamulin Ointment

[0065] Weigh lefamulin (0.5 g) and place it in a beaker. Add white vaseline and heat to promote dissolution. Then add polyoxyethylene stearate and propylene glycol in sequence. Heat and stir to melt. Stir until solidified to obtain lefamulin ointment.

[0066] Example 1B Preparation of Retapamulin Ointment

[0067] 1B) Prepare the ingredients according to the following formula:

[0068]

[0069] 2B) Preparation of Retapamulin Ointment

[0070] Weigh retapamulin (0.5 g) and place it in a beaker. Add white vaseline and heat to promote dissolution. Then add polyoxyethylene stearate and propylene glycol in sequence. Heat and stir to melt. Stir until solidified to obtain retapamulin ointment.

[0071] Example 2 Effects of Topical Administration of Lefamulin or Retapamulin on IMQ-Induced Mouse Psoriasis Model Example 2A Effects of Topical Administration of Lefamulin on IMQ-Induced Mouse Psoriasis Model

[0072] 2A.1 Test materials

[0073] 2A.1.1 Experimental animals and reagents

[0074] The experimental animals used were 7-8 week old male BALB / c mice, provided by Jicui Yaokang Animal Biotechnology Experimental Center, certificate number: SCXK(Su)2018-0008.

[0075] The experimental reagents used were IMQ cream (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.) and the lefamulin ointment prepared in Example 1A.

[0076] 2A.1.2 Experimental Methods

[0077] Twenty-four male BALB / c mice aged 7-8 weeks were randomly divided into two groups, with 6 mice in each group. The backs of the mice were shaved (area: 2 cm×3 cm) and they were raised normally. Three days later, different treatments were given to the shaved areas of the mice. The specific operations were as follows:

[0078] Modeling group (IMQ): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days;

[0079] Treatment group (IMQ+5% Lefamulin): 62.5 mg of IMQ / mouse / day was applied to the back skin of mice for 5 days; 12 hours after applying IMQ every day, an equal dose of 5% Lefamulin ointment prepared in Example 1A was applied.

[0080] The mice in the above groups were fed and drank normally, and the skin manifestations of the mice were observed every day. On the 6th day, the mice were killed and the skin lesions were obtained for (H&E) staining.

[0081] 2A.1.3 Experimental Results

[0082] The changes of the back skin and skin lesions of each group of mice on the 6th day were shown in HE staining (4X). Figure 1 As shown in A.

[0083] Depend on Figure 1 As shown in Figure A, compared with the blank control group, obvious erythema and scaling were observed on the back skin of mice in the modeling group (IMQ); HE staining showed obvious hyperkeratosis and parakeratosis of the epidermis, and a large number of inflammatory cells infiltrated in the superficial dermis. The treatment group (IMQ + 5% Lefamulin) showed that erythema and scaling were significantly alleviated after topical drug treatment.

[0084] Example 2B Effect of topical administration of retapamulin on IMQ-induced psoriasis model in mice

[0085] 2B.1. Test Materials

[0086] 2B.1.1 Experimental animals and reagents

[0087] The experimental animals used were 7-8 week old male BALB / c mice, provided by Jicui Yaokang Animal Biotechnology Experimental Center, certificate number: SCXK(Su)2018-0008.

[0088] The experimental reagents used were the retapamulin ointment prepared in Example 1B and IMQ cream (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.).

[0089] 2B.1.2 Experimental methods

[0090] Twenty-four male BALB / c mice aged 7-8 weeks were randomly divided into two groups, with 6 mice in each group. The backs of the mice were shaved (area: 2 cm×3 cm) and they were raised normally. Three days later, different treatments were given to the shaved areas of the mice. The specific operations were as follows:

[0091] Modeling group (IMQ): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days;

[0092] Treatment group (IMQ+5% Retapamulin): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days; 12 hours after applying IMQ every day, an equal dose of 5% Retapamulin ointment prepared in Example 1B was applied.

[0093] The mice in the above groups were fed and drank normally, and the skin manifestations of the mice were observed every day. On the 6th day, the mice were killed and the skin lesions were obtained for (H&E) staining.

[0094] 2B.1.3 Experimental Results

[0095] The changes of the back skin and skin lesions of each group of mice on the 6th day were shown in HE staining (4X). Figure 1 As shown in B.

[0096] Depend on Figure 1 Figure B shows that compared with the blank control group, obvious erythema and scaling were observed on the back skin of mice in the modeling group (IMQ); HE staining showed obvious hyperkeratosis and parakeratosis of the epidermis, and a large number of inflammatory cells infiltrated in the superficial dermis. The treatment group (IMQ + 5% Retapamulin) showed that erythema and scaling were significantly alleviated after topical drug treatment.

[0097] The experiment in Example 2 showed that in the IMQ-induced psoriasis mouse model, topical administration of famolin or retapamulin can alleviate IMQ-induced psoriasis-like dermatitis, and topical administration is more efficient and convenient in skin diseases.

[0098] Example 3 Effects of Lefamulin and Retapamulin on TNF-α-induced L929 cell death model

[0099] Example 3A Effect of Lefamulin on TNF-α-induced L929 cell death model

[0100] 3A.1 Test Materials

[0101] Mouse fibroblast L929 cell line in logarithmic growth phase was used for the experiment;

[0102] Human TNF-α was dissolved in purified water at a concentration of 10 ng / ml; lefamulin was dissolved in dimethyl sulfoxide to prepare solutions with concentrations of 10 μM, 30 μM, 50 μM, 70 μM and 100 μM, respectively; CCK8 cell death detection kit was purchased from MCE.

[0103] 3A.2 Experimental methods

[0104] Divided into 3 groups, the specific operations are as follows:

[0105] Negative control group: complete DMEM medium 100 μl / well, 4 replicate wells per 96-well plate;

[0106] Cell death group: 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with 4 replicates per 96-well plate;

[0107] Drug group: Lefamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added at five different concentrations of 10 μM, 30 μM, 50 μM, 70 μM and 100 μM, with 4 replicate wells for each concentration;

[0108] The 96-well plate was placed in a 37°C, 5% carbon dioxide incubator for 7 hours, and 10 μl / well of CCK-8 reagent was added to the 96-well plate and incubated in a 37°C, 5% carbon dioxide incubator for 2 hours. The optical density of each well was detected by a microplate reader at a wavelength of 450 nm, and the background OD value (complete culture medium plus CCK8 detection solution, no cells) was subtracted from the OD value of each test well to calculate the mean and standard deviation, and the calculated value = (drug group / cell death group) / (negative control / cell death group).

[0109] 3A.3 Experimental Results

[0110] After L929 cells were treated with 10 ng / ml TNF-α and 1 μg / ml actinomycin D for 7 hours, the cell viability was detected by CCK-8 method, and about 90% of L929 cells showed cell death. In the drug group, different concentrations of lefamulin could significantly inhibit TNF-α-induced L929 cell death, and the P value was calculated by two-tailed Student's t test (Figure 2 Figure 2A shown).

[0111] Example 3B Effect of Retapamulin on TNF-α-induced L929 cell death model

[0112] 3B.1. Test materials

[0113] Mouse fibroblast L929 cell line in logarithmic growth phase was used for the experiment;

[0114] Human TNF-α was dissolved in purified water at a concentration of 10 ng / ml; Retapamulin was dissolved in dimethyl sulfoxide to prepare solutions with concentrations of 10 μM, 30 μM, 50 μM, 70 μM and 100 μM, respectively; CCK8 cell death detection kit was purchased from MCE.

[0115] 3B.2 Experimental methods

[0116] Divided into 3 groups, the specific operations are as follows:

[0117] Negative control group: complete DMEM medium 100 μl / well, 4 replicate wells per 96-well plate;

[0118] Cell death group: 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with 4 replicates per 96-well plate;

[0119] Drug group: Retapamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added at five different concentrations of 10 μM, 30 μM, 50 μM, 70 μM and 100 μM, with 4 replicate wells for each concentration;

[0120] The 96-well plate was placed in a 37°C, 5% carbon dioxide incubator for 7 hours, and 10 μl / well of CCK-8 reagent was added to the 96-well plate and incubated in a 37°C, 5% carbon dioxide incubator for 2 hours. The optical density of each well was detected by a microplate reader at a wavelength of 450 nm, and the background OD value (complete culture medium plus CCK8 detection solution, no cells) was subtracted from the OD value of each test well to calculate the mean and standard deviation, and the calculated value = (drug group / cell death group) / (negative control / cell death group).

[0121] 3B.3 Experimental results

[0122] After L929 cells were treated with 10 ng / ml TNF-α and 1 μg / ml actinomycin D for 7 hours, the cell viability was detected by CCK-8 method, and about 90% of L929 cells showed cell death. In the drug group, different concentrations of retapamulin could significantly inhibit TNF-α-induced L929 cell death, and the P value was calculated by two-tailed Student's t test (Figure 2 Figure 2B shown).

[0123] It can be seen that the results of Example 3 show that in in vitro experiments, lefamulin or retapamulin can directly inhibit TNF-α-induced L929 cell death, so lefamulin or retapamulin can inhibit TNF-α and ultimately achieve a therapeutic effect on psoriasis.

[0124] Example 4 Using chemical proteomics to find that lefamulin interacts with hnRNP-U

[0125] 4.1 Experimental materials and methods

[0126] The human epidermal immortalized cell HaCaT cell line and mouse fibroblast L929 cell line in the logarithmic growth phase were used for the experiment, and a photo-crosslinking group (Lef-probe) was added to lefamulin. The molecular structure of the photo-crosslinking group (lef-probe) added to lefamulin is as follows Figure 3A shown.

[0127] The effect of Lef-probe on the TNF-α-induced L929 cell death model was detected using the same detection method as in Example 3. Figure 3C As shown, the grouping and steps of proteomic screening of lefamulin protein substrates are as follows:

[0128] 1) L929 or HaCaT cells were co-incubated with Lef-probe. To improve the specificity of mass spectrometry data, the experiment was divided into two pairs of combinations (e.g. Figure 3C As shown): ① UV irradiation and non-UV irradiation groups (calculation of UV enrichment intensity), ② UV irradiation, lefamulin and non-lefamulin groups (calculation of lefamulin competition intensity);

[0129] 2) Using click chemistry to couple the probe-target protein complex to biotin;

[0130] 3) Use streptavidin coupling to enrich the probe-target protein-biotin complex;

[0131] 4) Labeling of target proteins using tandem mass spectrometry;

[0132] 5) Liquid chromatography tandem mass spectrometry analysis.

[0133] 4.2 Experimental Results

[0134] like Figure 3B As shown in Figure 2, Lef-probe can inhibit TNF-α-induced cell death, indicating that the photocrosslinking group does not affect the biological function of lefamulin. In the chemical proteomics experiment, the experiment was repeated three times independently in two cell lines (such as Figure 3D As shown in FIG, after screening the potential protein substrates from L929 and HaCaT cells, 97 potential target proteins were obtained in the L929 cell line and 69 potential target proteins were obtained in the HaCaT cell line. The data of the two cell lines were cross-analyzed, as shown in FIG. Figure 3E As shown, the ratio of proteins interacting with lefamulin identified in L929 and HaCaT cells, among which the black spot with the largest area is hnRNP-U. Figure 3E In the figure, the Y-axis is the UV enrichment ratio (Ruv=UV irradiation mass spectrum abundance value / non-UV irradiation mass spectrum abundance value, Ruv>1.1 is positive), and the X-axis is the lefamulin competition ratio (Rcomp=Lef-probe group mass spectrum abundance value / lefamulin mass spectrum abundance value, Rcomp>1.1 is positive). The applicant found that lefamulin is most likely to interact with heterogeneous nuclear ribonucleoprotein-U (hnRNP-U). It can be seen that the results of Example 4 show that lefamulin interacts with hnRNP-U in in vitro experiments.

[0135] Example 5 Lefamulin can affect the function of hnRNP-U protein and thus inhibit the TNF-α pathway

[0136] 5.1 Test materials

[0137] The experiments were conducted using the immortalized human epidermal HaCaT cell line and the mouse fibroblast L929 cell line in the logarithmic growth phase.

[0138] Famolin was dissolved in dimethyl sulfoxide to prepare solutions with concentrations of 50 μM and 100 μM, respectively. The primary antibody for immunoblotting was purchased from Abcam: hnRNP-U (ab180952). The mouse hnRNP-U siRNA sequences were: GGAGCAAUAUAAAGAAGAATT and UUCUUCUUUUAUAUUGCUCCTT. siRNA transfection reagent (Lipofectamine TM 3000 kit) was purchased from ThermoFisher. CCK8 cell death detection kit was purchased from MCE. Human TNF-α was dissolved in purified water at a concentration of 10 ng / ml.

[0139] 5.2 Experimental methods

[0140] 5.2.1 Immunoblotting

[0141] Divided into 3 groups, the specific operations are as follows:

[0142] Blank control group (Control): Protein extracted from HaCaT cell line, i.e., protein extracted from HaCaT cells using RIPA lysis buffer was used as blank control;

[0143] 12-hour incubation lefamulin group (TNF-α+50μM Lefamulin): HaCaT cells were treated with 50μM lefamulin for 12 hours; HaCaT cell proteins were then extracted using RIPA lysis buffer;

[0144] 24-hour incubation lefamulin group (TNF-α+50μM Lefamulin): HaCaT cells were treated with 50μM lefamulin for 24 hours; HaCaT cell proteins were then extracted using RIPA lysis buffer;

[0145] At room temperature, 100 μl of RIPA lysis buffer was added to the cells. After lysis for 2 minutes, the liquid was collected into a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes in a high-speed centrifuge at 4°C. The supernatant was aspirated to obtain the protein sample. Equal amounts of protein samples were separated by electrophoresis using a constant voltage method of 150 V and 4-20% sodium dodecyl sulfate polyacrylamide gel (Bio-RAD) at room temperature; the protein was transferred to a PVDF membrane (Bio-RAD) using a semi-dry transfer method; the PVDF membrane was blocked with 5% skim milk powder for 1 hour at room temperature; the PVDF membrane was incubated with the primary antibody (i.e., the first antibody for immunoblotting) at 4°C overnight, and then incubated with the horseradish peroxidase secondary antibody (ABclonal) for 1 hour at room temperature. The chemiluminescent signal was detected and analyzed using the Gel-Doc XR imaging laboratory system (Bio-RAD).

[0146] 5.2.2 Cell death assay

[0147] Divided into 5 groups, the specific operations are as follows:

[0148] Negative control group: complete DMEM medium 100 μl / well, 4 replicate wells per 96-well plate;

[0149] Cell death group: 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with 4 replicates per 96-well plate;

[0150] hnRNP-U knockdown cell death group: cells were transfected with siRNA for 48 hours to knock down hnRNP-U expression, and 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with 4 replicates per 96-well plate;

[0151] Drug group: 50 μM lefamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α and 1 μg / ml actinomycin D were added, with 4 replicate wells for each concentration;

[0152] hnRNP-U knockdown drug group: cells were transfected with siRNA for 48 hours to knock down hnRNP-U expression, and lefamulin (dissolved in DMSO, DMSO concentration ≤ 0.1%), 10 ng / ml TNF-α, and 1 μg / ml actinomycin D were added at a concentration of 50 μM, with 4 replicates for each concentration;

[0153] The cell death detection method is the same as in Example 3.

[0154] 5.3 Experimental Results

[0155] like Figure 4A As shown in the figure, in the drug-treated group, lefamulin could significantly inhibit the expression of hnRNP-U in HaCaT cells at both 12 hours and 24 hours. Figure 4B As shown, using specific siRNA to knock down hnRNP-U, it was found that knocking down hnRNP-U led to a weakened function of lefamulin in inhibiting the TNF-α pathway in the L929 cell death model.

[0156] like Figure 4A and 4B As shown, the results of Example 5 indicate that lefamulin can directly inhibit the expression of hnRNP-U in vitro experiments. Therefore, lefamulin may inhibit TNF-α through hnRNP-U and ultimately achieve a therapeutic effect on psoriasis.

[0157] Example 6hnRNP-U is associated with psoriasis disease activity

[0158] 6.1 Experimental animals, psoriasis lesions, and reagents

[0159] C57 mice, male, 7-8 weeks old, were provided by Jicui Yaokang Animal Biotechnology Experimental Center, certificate number: SCXK(Su)2018-0008.

[0160] The patient's skin lesions were provided by the Dermatology Hospital of Chinese Academy of Medical Sciences, and the experiment was approved by the Ethics Committee of the Dermatology Hospital of Chinese Academy of Medical Sciences.

[0161] IMQ cream (purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.), hnRNP-U antibody (purchased from Abcam).

[0162] 6.2 Experimental methods

[0163] 6.2.1 Animal experiments

[0164] Six male C57 mice aged 7-8 weeks were randomly divided into two groups, with 6 mice in each group. The backs of the mice were shaved (area: 2cm×3cm) and they were raised normally. Three days later, different treatments were given to the shaved areas of the mice. The specific operations were as follows:

[0165] Blank control group (Control): 62.5 mg of vaseline was applied to the back skin of mice per day for 5 days;

[0166] Modeling group (IMQ): IMQ 62.5 mg / mouse / day was applied to the back skin of mice for 5 days;

[0167] The mice in the above groups were fed and watered normally, and the skin manifestations of the mice were observed every day. The mice were killed on the 6th day and the skin lesions of the mice were embedded in paraffin and stained with HE. Paraffin sections with a thickness of 5 μm were cut for hnRNP-U immunohistochemical staining, and the primary antibody was used (Abcam, ab180952, 1:100 dilution). Pictures were taken under a 4X field of view, and the number of hnRNP-U positive cells was counted under a 40X field of view using the software Image J, and the average value of hnRNP-U positive cells in 3 random fields of view for each mouse was calculated.

[0168] 6.2.2 Experiments with human tissue specimens

[0169] Human psoriasis lesions were obtained surgically, embedded in paraffin and stained with HE. 5 μm thick paraffin sections were cut for hnRNP-U immunohistochemical staining, and the primary antibody used was (Abcam, ab180952, 1:100 dilution).

[0170] 6.3 Experimental Results

[0171] like Figure 5A to Figure 5C As shown in the figure, in the mouse experiment, the hnRNP-U immunohistochemical staining in the skin modeling area was significantly enhanced compared with the blank control group. In the human tissue specimen experiment, the expression of hnRNP-U in the lesional area was significantly higher than that in the non-lesional area.

[0172] It can be seen that the experiment in Example 6 shows that hnRNP-U is related to the disease activity of psoriasis, so lefamulin can inhibit TNF-α through hnRNP-U and ultimately achieve a therapeutic effect on psoriasis.

[0173] It can be seen that the present invention firstly models psoriasis in mice by using imiquimod, and psoriatic dermatitis appears on the back skin of mice. Psoriatic dermatitis is treated by using 5% lefamulin and retapamulin ointment, and it can be found that both drugs can treat imiquimod-induced psoriatic dermatitis. In addition, the present invention uses TNF-α to model cell death in L929 cell line, and the cell death effect of L929 induced by TNF-α is inhibited by lefamulin and retapamulin, and the inhibition efficiency is dose-dependent. In addition, the present invention adds a photocrosslinking group to lefamulin, and through chemical proteomics screening, it is found that hnRNP-U can interact with lefamulin. On this basis, in L929 cells, hnRNP-U in cells is knocked down using hnRNP-U specific siRNA, and it is found that the function of lefamulin to inhibit TNF-α-induced L929 cell death is weakened after knocking down hnRNP-U. In HaCaT cells, lefamulin is used for co-incubation, and it is found that the expression of hnRNP-U in HaCaT cells decreases. The above experiments show that lefamulin can inhibit the TNF-α pathway through hnRNP-U, and in the prior art, no inhibitors or agonists for hnRNP-U have been reported. Finally, in psoriasis-like lesions of mice and lesions of psoriasis patients, it was found that the expression of hnRNP-U was correlated with the severity of the disease, which indicates that lefamulin can achieve the purpose of treating psoriasis by inhibiting hnRNP-U.

[0174] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a compound in the preparation of a drug for treating psoriasis, characterized in that: The compound is lefamulin, retapamulin or a derivative thereof, Wherein, the structural formula of lefamulin is as follows: The structural formula of retapamulin is shown below:

2. The use according to claim 1, characterized in that The derivatives are in the form of salts and / or solvates.

3. The use according to claim 1, characterized in that The derivatives of lefamulin are in the form of lefamulin acetate and lefamulin hydrochloride.

4. The use according to claim 1, characterized in that The psoriasis is psoriasis vulgaris.

5. The use according to claim 1, characterized in that The medicine contains therapeutically effective amounts of lefamulin, retapamulin and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The therapeutically effective amount is 1% to 10%.

7. The use according to claim 5, characterized in that The pharmaceutically acceptable carrier includes one or more of a diluent, a solubilizer, a latent solvent, a disintegrant, a dispersant, a lubricant, a flavoring agent, an antioxidant, a binder, an absorbent, a wetting agent, a buffer and a cross-linking agent.

8. The use according to claim 1, characterized in that The drug is prepared into a pharmaceutically acceptable dosage form, wherein the dosage form includes pills, tablets, powders, capsules, granules, powders, pellets, drops, patches, tinctures, pastes, lotions, sprays, injections, suspensions, creams, frosts, ointments, gels or suppositories.

9. An ointment for treating psoriasis, wherein the ointment comprises lefamulin or retapamulin, in, The structural formula of lefamulin is shown below: The structural formula of retapamulin is shown below:

10. The ointment according to claim 9, wherein the composition of the ointment is as follows:

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